US4229441A - Immunologic adjuvant - Google Patents

Immunologic adjuvant Download PDF

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US4229441A
US4229441A US05/965,559 US96555978A US4229441A US 4229441 A US4229441 A US 4229441A US 96555978 A US96555978 A US 96555978A US 4229441 A US4229441 A US 4229441A
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thio
cholesten
yloxy
hexyl
acetyl
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US05/965,559
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Robert L. Bugianesi
Mitree M. Ponpipom
Tsung-Ying Shen
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Merck and Co Inc
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Merck and Co Inc
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Priority to US05/965,559 priority Critical patent/US4229441A/en
Priority to AU53009/79A priority patent/AU5300979A/en
Priority to CA000340239A priority patent/CA1147722A/en
Priority to EP79400932A priority patent/EP0012083A1/en
Priority to JP15447979A priority patent/JPS5589298A/en
Priority to DK509579A priority patent/DK509579A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J31/00—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring
    • C07J31/006—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring not covered by C07J31/003
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to an immunologic adjuvant and, more particularly to novel glycolipid immunologic adjuvant and to improved vaccine formulations containing a novel glycolipid immunologic adjuvant.
  • the vaccines utilized at the present time are "fluid vaccines.”
  • the term "fluid vaccine” designates a suspension of an immunogenic or desensitizing agent in water or in a medium comprising a single, aqueous, liquid phase.
  • the principal purpose for employment of an immunologic adjuvant is to achieve a more durable immunity of a higher level employing a smaller antigenic mass in a fewer number of doses than could be achieved by administration of the equivalent aqueous antigen. It may be noted that development of an immunologically satisfactory and pharmacologically acceptable adjuvant is a prime essential for the preparation of workable multivalent killed virus vaccines which are effective and practical in the prevention of viral, bacterial, mycoplasmal or rickettsial diseases.
  • Glycolipid compounds of the formulae ##STR3## wherein R is ##STR4## are useful immunologic adjuvants in vaccines.
  • glycolipid compounds of the present invention which are useful as immunologic adjuvants are prepared starting from per-O-acetyl-1-thioglycopyranose and 6-(5-cholesten-3 ⁇ -yloxy)hexyl iodide.
  • Equimolar amounts of the foregoing compounds may be condensed in an inert, non-polar solvent such as a halogenated solvent, e.g., dichloromethane or chloroform in the presence of a base such as, e.g., triethylamine, 1,5-diazabicyclo[5.4.0]-undec-5-ene, or 1,5-diazabicyclo[4.3.0]-non-5-ene.
  • the reaction may be carried out at from about 10° to about 30° C. under an inert atmosphere. Depending upon the base employed the reaction may take from about half an hour to about a few days. Thus, when employing 1,5-diazabicyclo[5.4.0]-undec-5-ene, or 1,5-diazabicyclo[4.3.0]-non-5-ene the reaction is usually completed in from about 0.5 to about 3 hours, while when employing triethylamine the reaction is usually completed in from about 1 to about 3 days.
  • the solution is washed with water and dried if the solvent was a halogenated solvent, or if the solvent was tetrahydrofuran, the solution is evaporated to dryness and the residue is partitioned between dichloromethane and water.
  • the dried solution is concentrated to a syrup which is put on a silica gel column and eluted with chloroform followed by 1-2% ethanol in chloroform.
  • the desired fractions are pooled and evaporated to give the blocked product 6-(5-cholesten-3 ⁇ -yloxy)hexyl per-O-acetyl-1-thio-glycopyranoside which is deblocked by basic ion exchange treatment or sodium methoxide in methanol to give the desired final product.
  • novel adjuvants of the invention may be employed to potentiate the antibody response of antigenic materials.
  • antigenic materials include one or more non-viable immunogenic or desensitizing (anti-allergic) agents of bacterial, viral or other origin.
  • the antigen component of the products of the invention may consist of a dried powder, an aqueous solution, an aqueous suspension and the like, including mixtures of the same, containing a non-viable immunogenic or desensitizing agent or agents.
  • the aqueous phase may conveniently be comprised of the antigenic material in a parenterally acceptable liquid.
  • the aqueous phase may be in the form of a vaccine in which the antigen is dissolved in a balanced salt solution, physiological saline solution, phosphate buffered saline solution, tissue culture fluids or other media in which the organism may have been grown.
  • the aqueous phase also may contain preservatives and/or substances conventionally incorporated in vaccine preparations.
  • the adjuvant emulsions of the invention may be prepared employing techniques well known to the art.
  • the antigen may be in the form of purified or partially purified antigen derived from bacteria, viruses, rickettsia or their products, or extracts of bacteria, viruses, or rickettsia, or the antigen may be an allergen such as pollens, dusts, danders, or extracts of the same or the antigen may be in the form of a poison or a venom derived from poisonous insects or reptiles.
  • the antigens will be in the form in which their toxic or virulent properties have been reduced or destroyed and which when introduced into a suitable host will either induce active immunity by the production therein of antibodies against the specific microorganisms, extract or products of microorganisms used in the preparation of the antigen, or, in the case of allergens, they will aid in alleviating the symptoms of the allergy due to the specific allergen.
  • the antigens can be used either singly or in combination for example, multiple bacterial antigens, multiple viral antigens, multiple mycoplasmal antigens, multiple rickettsial antigens, multiple bacterial or viral toxoids, multiple allergens or combinations of any of the foregoing products can be combined in the aqueous phase of the adjuvant composition of this invention.
  • Antigens of particular importance are derived from bacteria such as B. pertussis, Leptospira pomona and icterohaemorrhagiae, S. typhosa, S. paratyphi A and B, C. diphtheriae, C. tetani, C. botulinum, C. perfringens, C.
  • feseri and other gas gangrene bacteria B. anthracis, P, pestis, P. multocida, V. cholerae, Neisseria meningitidis, N, gonorrheae, Hemophilus influenzae, Treponema pollidum, and the like; from viruses as polio virus (multiple types), adeno virus (multiple types), parainfluenza virus (multiple types), measles, mumps, respiratory syncytial virus, influenza (various types), shipping fever virus (SF 4 ), Western and Eastern equine encephalomyelitis, Japanese B.
  • encephalomyelitis Russian Spring Summer encephalomyelitis, hog cholera virus, Newcastle disease virus, fowl pox, rabies, feline and canine distemper and the like viruses, from rickettsiae as epidemic and endemic typhus or other members of the spotted fever group, from various spider and snake venoms or any of the known allergens for example from ragweed, house dust, pollen extracts, grass pollens and the like.
  • 2,3,4-Tri-O-acetyl-1-thio- ⁇ -L-fucopyranose (10 mmol) is treated with 6-(5-cholesten-3 ⁇ -yloxy)hexyl iodide (10 mmol) in dichloromethane (30 ml) containing triethylamine (10 mmol).
  • the reaction takes place in 1 day at room temperature under nitrogen.
  • the resulting solution is washed with distilled water (20 ml) and dried with anhydrous sodium sulfate.
  • the filtered solution is concentrated to form a syrup which is put on a silica gel column and eluted with chloroform followed by 1.0% ethanol in chloroform.
  • the fractions containing the title compound, as determined by thin layer chromatography, are pooled and evaporated to give the title compound in 61% yield [ ⁇ ] D -4° (c 1.5, chloroform).
  • the blocked product from Step A is stirred with a basic ion exchange resin, Bio-Rad AG 1-X2 (OH), in ethanol-tetrahydrofuran or sodium methoxide in methanol to give the title compound as needles, yield 80%, m.p. 110°-112° (ethyl acetate), [ ⁇ ] D -11° (c 1.43, chloroform).
  • a basic ion exchange resin Bio-Rad AG 1-X2 (OH)
  • ethanol-tetrahydrofuran or sodium methoxide in methanol to give the title compound as needles, yield 80%, m.p. 110°-112° (ethyl acetate), [ ⁇ ] D -11° (c 1.43, chloroform).
  • Example 1A The product of Example 1A is repeated except using 2,3,4,6-tetra-O-acetyl-1-thio- ⁇ -D-glucopyranose (10 mmol) in lieu of 2,3,4-tri-O-acetyl-1-thio- ⁇ -L-fucopyranose and using 1,5-diazabicyclo[5.4.0]-undec-5-ene (10 mmol) in lieu of triethylamine.
  • the reaction takes place in 2 hours at room temperature under nitrogen.
  • the title compound is obtained in 86% yield, m.p. 101°-102.5° (methanol), [ ⁇ ] D -36° (c 1.59, chloroform).
  • Example 1 The procedure of Example 1 is repeated except using 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio ⁇ -D-glucopyranose (10 mmol) in lieu of 2,3,4-tri-O-acetyl-1-thio- ⁇ -L-fucopyranose and using tetrahydrofuran (30 ml) in lieu of dichloromethane, and upon completion of the reaction, the mixture is evaporated to dryness and the residue partioned between dichloromethane (30 ml) and water (20 ml) and dried before forming the syrup which is put on the silica gel column. The title compound is obtained in 90% yield, m.p. 176°-179°, [ ⁇ ] D -43° (c 1.5, chloroform).
  • Example 2A The procedure of Example 2A is repeated except using 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio- ⁇ -D-galactopyranose (10 mmol) in lieu of 2,3,4,6-tetra-O-acetyl-1-thio- ⁇ -D-glucopyranose.
  • the title compound is obtained as a crystalline material, yield 43%, m.p. 130°-133° (ethanol), [ ⁇ ] D -37° (c 1.5, chloroform).
  • Example 2A The procedure of Example 2A is repeated except using 2,3,4-tri-O-acetyl-1-thio- ⁇ -xylopyranose in lieu of 2,3,4,6-tetra-O-acetyl-1-thio- ⁇ -D-glucopyranose.
  • the title compound is obtained in 65% yield, m.p. 106°-108° (methanol), [ ⁇ ] D -56° (C 1.55, chloroform).
  • Example 2A The procedure of Example 2A is repeated except using 2,3,4-tri-O-acetyl-1-thio- ⁇ -L-arabinopyranose (10 mmol) in lieu of 2,3,4,6-tetra-O-acetyl-1-thio- ⁇ -D-glucopyranose.
  • the title compound is obtained in 64% yield, [ ⁇ ] D -22° (c 1.88, chloroform).
  • Example 2A The procedure of Example 2A is repeated except using hepta-O-acetyl-1-thio- ⁇ -lactose (10 mmol) in lieu of 2,3,4,6-tetra-O-acetyl-1-thio- ⁇ -D-glucopyranose.
  • the title compound is obtained in 46% yield, [ ⁇ ] D -22° (c 1.55 chloroform).
  • Thiourea (7.5 g) is added to a solution of 6-(5-cholesten-3 ⁇ -yloxy)hexyl iodide (15 g, 25.3 mmol) in tetrahydrofuran (200 ml) and the mixture is heated with stirring under reflux for 6 hours. The solution is concentrated to a residue which is triturated with anhydrous ether. The solid is filtered and dissolved in chloroform (100 ml). This solution is added to a solution of potassium metabisulfite (15 g) in water (100 ml). The mixture is heated under reflux in a nitrogen atmosphere for 20 minutes. The organic layer is washed with water, dried, and concentrated to dryness.
  • a solution of the blocked glycolipid (46 mg) in dry methanol (8 ml) containing sodium methoxide (3 mg) is kept for 3 hours at room temperature.
  • the medium is adjusted to pH 9 by gradual addtion of 2.5 N sodium hydroxide (10 drops).
  • the suspension is stirred for 16 hours and tetrahydrofuran (20 ml) is added to dissolve the precipitates.
  • the solution is de-ionized with acidic resin, filtered, and concentrated to dryness. The residue is triturated with ethyl ether-petroleum ether to give the title compound (30 mg, 80% yield).
  • An aqueous suspension of the final product of Example 1 in phosphate buffered saline (PBS) is sterile filtered and added in levels of 0.005 mg and 0.05 mg to 2 samples of bivalent whole influenza vaccine (A Victoria and B Hong Kong strains). Similar adjuvant vaccine preparations are prepared using the final products of examples 2-8.
  • PBS phosphate buffered saline

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Abstract

Glycolipid compounds of the formulae ##STR1## wherein R is ##STR2## are useful immunologic adjuvants in vaccines.

Description

BACKGROUND OF THE INVENTION
The present invention relates to an immunologic adjuvant and, more particularly to novel glycolipid immunologic adjuvant and to improved vaccine formulations containing a novel glycolipid immunologic adjuvant.
Broadly considered, the vaccines utilized at the present time are "fluid vaccines." The term "fluid vaccine" designates a suspension of an immunogenic or desensitizing agent in water or in a medium comprising a single, aqueous, liquid phase. The principal purpose for employment of an immunologic adjuvant is to achieve a more durable immunity of a higher level employing a smaller antigenic mass in a fewer number of doses than could be achieved by administration of the equivalent aqueous antigen. It may be noted that development of an immunologically satisfactory and pharmacologically acceptable adjuvant is a prime essential for the preparation of workable multivalent killed virus vaccines which are effective and practical in the prevention of viral, bacterial, mycoplasmal or rickettsial diseases.
OBJECTS OF THE INVENTION
It is an object of the present invention to provide new glycolipid compounds. Another object is to provide methods for preparing these glycolipid compounds. A further object is to provide vaccine compositions containing these glycolipid compounds. These and other objects of the present invention will be apparent from the following description.
SUMMARY OF THE INVENTION
Glycolipid compounds of the formulae ##STR3## wherein R is ##STR4## are useful immunologic adjuvants in vaccines.
DETAILED DESCRIPTION
The glycolipid compounds of the present invention which are useful as immunologic adjuvants are prepared starting from per-O-acetyl-1-thioglycopyranose and 6-(5-cholesten-3β-yloxy)hexyl iodide. Equimolar amounts of the foregoing compounds may be condensed in an inert, non-polar solvent such as a halogenated solvent, e.g., dichloromethane or chloroform in the presence of a base such as, e.g., triethylamine, 1,5-diazabicyclo[5.4.0]-undec-5-ene, or 1,5-diazabicyclo[4.3.0]-non-5-ene. The reaction may be carried out at from about 10° to about 30° C. under an inert atmosphere. Depending upon the base employed the reaction may take from about half an hour to about a few days. Thus, when employing 1,5-diazabicyclo[5.4.0]-undec-5-ene, or 1,5-diazabicyclo[4.3.0]-non-5-ene the reaction is usually completed in from about 0.5 to about 3 hours, while when employing triethylamine the reaction is usually completed in from about 1 to about 3 days. Following the reaction the solution is washed with water and dried if the solvent was a halogenated solvent, or if the solvent was tetrahydrofuran, the solution is evaporated to dryness and the residue is partitioned between dichloromethane and water. The dried solution is concentrated to a syrup which is put on a silica gel column and eluted with chloroform followed by 1-2% ethanol in chloroform. The desired fractions are pooled and evaporated to give the blocked product 6-(5-cholesten-3β-yloxy)hexyl per-O-acetyl-1-thio-glycopyranoside which is deblocked by basic ion exchange treatment or sodium methoxide in methanol to give the desired final product.
The novel adjuvants of the invention may be employed to potentiate the antibody response of antigenic materials. The term "antigen" and "antigenic material" which are used interchangeably herein include one or more non-viable immunogenic or desensitizing (anti-allergic) agents of bacterial, viral or other origin. The antigen component of the products of the invention may consist of a dried powder, an aqueous solution, an aqueous suspension and the like, including mixtures of the same, containing a non-viable immunogenic or desensitizing agent or agents.
The aqueous phase may conveniently be comprised of the antigenic material in a parenterally acceptable liquid. For example, the aqueous phase may be in the form of a vaccine in which the antigen is dissolved in a balanced salt solution, physiological saline solution, phosphate buffered saline solution, tissue culture fluids or other media in which the organism may have been grown. The aqueous phase also may contain preservatives and/or substances conventionally incorporated in vaccine preparations. The adjuvant emulsions of the invention may be prepared employing techniques well known to the art.
The antigen may be in the form of purified or partially purified antigen derived from bacteria, viruses, rickettsia or their products, or extracts of bacteria, viruses, or rickettsia, or the antigen may be an allergen such as pollens, dusts, danders, or extracts of the same or the antigen may be in the form of a poison or a venom derived from poisonous insects or reptiles. In all cases the antigens will be in the form in which their toxic or virulent properties have been reduced or destroyed and which when introduced into a suitable host will either induce active immunity by the production therein of antibodies against the specific microorganisms, extract or products of microorganisms used in the preparation of the antigen, or, in the case of allergens, they will aid in alleviating the symptoms of the allergy due to the specific allergen. The antigens can be used either singly or in combination for example, multiple bacterial antigens, multiple viral antigens, multiple mycoplasmal antigens, multiple rickettsial antigens, multiple bacterial or viral toxoids, multiple allergens or combinations of any of the foregoing products can be combined in the aqueous phase of the adjuvant composition of this invention. Antigens of particular importance are derived from bacteria such as B. pertussis, Leptospira pomona and icterohaemorrhagiae, S. typhosa, S. paratyphi A and B, C. diphtheriae, C. tetani, C. botulinum, C. perfringens, C. feseri and other gas gangrene bacteria, B. anthracis, P, pestis, P. multocida, V. cholerae, Neisseria meningitidis, N, gonorrheae, Hemophilus influenzae, Treponema pollidum, and the like; from viruses as polio virus (multiple types), adeno virus (multiple types), parainfluenza virus (multiple types), measles, mumps, respiratory syncytial virus, influenza (various types), shipping fever virus (SF4), Western and Eastern equine encephalomyelitis, Japanese B. encephalomyelitis, Russian Spring Summer encephalomyelitis, hog cholera virus, Newcastle disease virus, fowl pox, rabies, feline and canine distemper and the like viruses, from rickettsiae as epidemic and endemic typhus or other members of the spotted fever group, from various spider and snake venoms or any of the known allergens for example from ragweed, house dust, pollen extracts, grass pollens and the like.
The following examples illustrate the present invention without, however, limiting the same thereto. All temperatures are expressed in degrees celsius.
EXAMPLE 1 6-(5-Cholesten-3β-yloxy)hexyl 1-thio-β-L-fucopyranoside A. 6-(5-Cholesten-3β-yloxy)hexyl 2,3,4-tri-O-acetyl 1-thio-β-L-fucopyranoside
2,3,4-Tri-O-acetyl-1-thio-β-L-fucopyranose (10 mmol) is treated with 6-(5-cholesten-3β-yloxy)hexyl iodide (10 mmol) in dichloromethane (30 ml) containing triethylamine (10 mmol). The reaction takes place in 1 day at room temperature under nitrogen. The resulting solution is washed with distilled water (20 ml) and dried with anhydrous sodium sulfate. The filtered solution is concentrated to form a syrup which is put on a silica gel column and eluted with chloroform followed by 1.0% ethanol in chloroform. The fractions containing the title compound, as determined by thin layer chromatography, are pooled and evaporated to give the title compound in 61% yield [α]D -4° (c 1.5, chloroform).
B. 6-(5-Cholesten-3β-yloxy)hexyl 1-thio-β-L-fucopyranoside
The blocked product from Step A is stirred with a basic ion exchange resin, Bio-Rad AG 1-X2 (OH), in ethanol-tetrahydrofuran or sodium methoxide in methanol to give the title compound as needles, yield 80%, m.p. 110°-112° (ethyl acetate), [α]D -11° (c 1.43, chloroform).
EXAMPLE 2 6-(5-Cholesten-3β-yloxy)hexyl 1-thio-β-D-glucopyranoside A. 6-(5-Cholesten-3β-yloxy)hexyl 2,3,4,6-tetra-Oacetyl-1-thio-β-D-glucopyranoside
The product of Example 1A is repeated except using 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranose (10 mmol) in lieu of 2,3,4-tri-O-acetyl-1-thio-β-L-fucopyranose and using 1,5-diazabicyclo[5.4.0]-undec-5-ene (10 mmol) in lieu of triethylamine. The reaction takes place in 2 hours at room temperature under nitrogen. The title compound is obtained in 86% yield, m.p. 101°-102.5° (methanol), [α]D -36° (c 1.59, chloroform).
B. 6-(5-Cholesten-3β-yloxy)hexyl 1-thio-β-D-glucopyranoside
The title compound is obtained following the procedure of Example 1B, yield 62%, m.p. 110° (aqueous isopropanol), [α]D -41° (c 1.07, chloroform), Rf 0.27 (chloroform-methanol, 9:1).
EXAMPLE 3 6-(5-Cholesten-3β-yloxy)hexyl 2-acetamido-2-deoxy-1-thioβ-D-glucopyranoside A. 6-(5-Cholesten-3β-yloxy)hexyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-β-D-glucopyranoside
The procedure of Example 1 is repeated except using 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thioβ-D-glucopyranose (10 mmol) in lieu of 2,3,4-tri-O-acetyl-1-thio-β-L-fucopyranose and using tetrahydrofuran (30 ml) in lieu of dichloromethane, and upon completion of the reaction, the mixture is evaporated to dryness and the residue partioned between dichloromethane (30 ml) and water (20 ml) and dried before forming the syrup which is put on the silica gel column. The title compound is obtained in 90% yield, m.p. 176°-179°, [α]D -43° (c 1.5, chloroform).
B. 6-(5-Cholesten-3β-yloxy)hexyl 2-acetamido-2-deoxy-1-thio-β-D-glucopyranoside
The title compound is obtained following the procedure of Example 1B, yield 77%, m.p. 183°-187° (dec) (methanol), [α]D -36° (c 1.5, dimethylsulfoxide), Rf 0.48 (chloroform-methanol-water (80:20:2).
EXAMPLE 4 6-(5-Cholesten-3β-yloxy)hexyl 2-acetamido-2-deoxy-1-thio-β-D-galactopyranoside A. 6-(5-Cholesten-3β-yloxy)hexyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-β-D-galactopyranoside
The procedure of Example 2A is repeated except using 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-β-D-galactopyranose (10 mmol) in lieu of 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranose. The title compound is obtained as a crystalline material, yield 43%, m.p. 130°-133° (ethanol), [α]D -37° (c 1.5, chloroform).
B. 6-(5-Cholesten-3β-yloxy)hexyl 2-acetamido-2-deoxy1-thio-β-D-galactopyranoside
The title compound is obtained folllowing the procedure of Example 1B, yield 85%, m.p. 241°-243°, [α]D -35° (c 1.5, N,N-dimethylformamide).
EXAMPLE 5 6-(5-Cholesten-3β-yloxy)hexyl 1-thio-β-D-xylopyranoside A. 6-(5-Cholesten-3β-yloxy)hexyl 2,3,4-tri-O-acetyl-1thio-β-D-xylopyranoside
The procedure of Example 2A is repeated except using 2,3,4-tri-O-acetyl-1-thio-β-xylopyranose in lieu of 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranose. The title compound is obtained in 65% yield, m.p. 106°-108° (methanol), [α]D -56° (C 1.55, chloroform).
B. 6-(5-Cholesten-3β-yloxy)hexyl 1-thio-β-D-xylopyranoside
The title compound is obtained as a crystalline material following the procedure of Example 1B, m.p. 115° (methanol), [α]D -45° (c 1.47,
EXAMPLE 6 6-(5-Cholesten-3β-yloxy)hexyl 1-thio-α-L-arabinopyranoside A. 6-(5-Cholesten-3β-yloxy)hexyl 2,3,4-tri-O-acetyl1-thio-α-L-arabinopyranoside
The procedure of Example 2A is repeated except using 2,3,4-tri-O-acetyl-1-thio-α-L-arabinopyranose (10 mmol) in lieu of 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranose. The title compound is obtained in 64% yield, [α]D -22° (c 1.88, chloroform).
B. 6-(5-Cholesten-3β-yloxy)hexyl 1-thio-α-L-arabinopyranoside
The title compound is obtained following the procedure of Example 1B, yield 83%, [α]d -18° (c 2.0, chloroform).
EXAMPLE 7 6-(5-Cholesten-3β-yloxy)hexyl 1-thio-β-lactoside A. 6-(5-Cholesten-3β-yloxy)hexyl hepta-O-acetyl-1-thio-β-lactoside
The procedure of Example 2A is repeated except using hepta-O-acetyl-1-thio-β-lactose (10 mmol) in lieu of 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranose. The title compound is obtained in 46% yield, [α]D -22° (c 1.55 chloroform).
B. 6-(5-Cholesten-3β-yloxy)hexyl 1-thio-β-lactoside
The title compound is obtained as a crystalline material following the procedure of Example 1B, m.p. 189°-192° (aqueous isopropanol), [α]D -21° (c 1.63, dimethylsulfoxide), Rf 0.26 (chloroform-methanol-water, 80:20:2).
EXAMPLE 8 2-S-[6-(5-Cholesten-3β-yloxy)hexyl]-2-thio-β-D-N-acetylneuraminic acid A. 6-(5-Cholesten-3β-yloxy)-hexane-1-thiol
Thiourea (7.5 g) is added to a solution of 6-(5-cholesten-3β-yloxy)hexyl iodide (15 g, 25.3 mmol) in tetrahydrofuran (200 ml) and the mixture is heated with stirring under reflux for 6 hours. The solution is concentrated to a residue which is triturated with anhydrous ether. The solid is filtered and dissolved in chloroform (100 ml). This solution is added to a solution of potassium metabisulfite (15 g) in water (100 ml). The mixture is heated under reflux in a nitrogen atmosphere for 20 minutes. The organic layer is washed with water, dried, and concentrated to dryness. The crude material is put on a silica gel column and eluted with 5% ethyl ether in peteroleum ether. The dried fractions are pooled and concentrated to give the title compound (11 g, 86% yield), m.p. 89°-90°.
B. Methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-2-S-[6-(5-cholesten-3β-yloxy)hexyl]-2-thio-D-neuraminate
Boron trifluoride etherate (700 μl, 5.5 mmol) is added to a solution of methyl 2,4,7,8,9-penta-O-acetyl-N-acetyl-D-neuraminate (1.01 g, 1.96 mmol) and 6-(5-cholesten-3β-yloxy)-hexane-1-thiol (0.98 g, 1.96 mmol) in dry chloroform (5 ml). The mixture is stirred under nitrogen for 5 hours at room temperature, and washed with aqueous sodium bicarbonate and water. The dried solution is concentrated to a residue which is put on a silica gel column and eluted with chloroform-ethyl ether-methanol (31:10:1). The β-anomer is isolated as a glass (400 mg), [α]D -38.5° (c 1.5, chloroform).
C. 2-S-[6-(5-Cholesten-3β-yloxy)hexyl]-2-thio-β-D-N-acetylneuraminic acid
A solution of the blocked glycolipid (46 mg) in dry methanol (8 ml) containing sodium methoxide (3 mg) is kept for 3 hours at room temperature. The medium is adjusted to pH 9 by gradual addtion of 2.5 N sodium hydroxide (10 drops). The suspension is stirred for 16 hours and tetrahydrofuran (20 ml) is added to dissolve the precipitates. The solution is de-ionized with acidic resin, filtered, and concentrated to dryness. The residue is triturated with ethyl ether-petroleum ether to give the title compound (30 mg, 80% yield).
EXAMPLE 9
An aqueous suspension of the final product of Example 1 in phosphate buffered saline (PBS) is sterile filtered and added in levels of 0.005 mg and 0.05 mg to 2 samples of bivalent whole influenza vaccine (A Victoria and B Hong Kong strains). Similar adjuvant vaccine preparations are prepared using the final products of examples 2-8.

Claims (4)

What is claimed is:
1. A compound selected from the group consisting of:
6-(5-cholesten-3β-yloxy)hexyl 1-thio-β-L-fucopyranoside;
6-(5-cholesten-3β-yloxy)hexyl 1-thio-β-D-glucopyranoside;
6-(5-cholesten-3β-yloxy)hexyl 2-acetamido-2-deoxy-1-thio-β-D-glucopyranoside;
6-(5-cholesten-3β-yloxy)hexyl 2-acetamido-2-deoxy-1-thio-β-D-galactopyranoside;
6-(5-cholesten-3β-yloxy)hexyl 1-thio-β-D-xylopyranoside;
6-(5-cholesten-3β-yloxy)hexyl 1-thio-α-L-arabinopyranoside;
6-(5-cholesten-3β-yloxy)hexyl 1-thio-β-lactoside, and
2-S-[6-(5-cholesten-3β-yloxy)hexyl]-2-thio-β-D-N-acetylneuraminic acid.
2. An intermediate for a compound of claim 1 selected from the group consisting of
6-(5-cholesten-3β-yloxy)hexyl 2,3,4-tri-O-acetyl-1-thio-β-L-fucopyranoside;
6-(5-cholesten-3β-yloxy)hexyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranoside;
6-(5-cholesten-3β-yloxy)hexyl 2-acetamido-3,4,6,-tri-O-acetyl-2-deoxy-1-thio-β-D-glucopyranoside;
6-(5-cholesten-3β-yloxy)hexyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-β-D-galactopyranoside;
6-(5-cholesten-3β-yloxy)hexyl 2,3,4-tri-O-acetyl-1-thio-β-D-xylopyranoside;
6-(5-cholesten-3β-yloxy)hexyl 2,3,4-tri-O-acetyl-1-thio-α-L-arabinopyranoside;
6-(5-cholesten-3β-yloxy)hexyl hepta-O-acetyl-1-thio-β-lactoside;
6-(5-cholesten-3β-yloxy)-hexane-1-thiol, and
Methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-2-S-[6-(5-cholesten-3β-yloxy)hexyl]2-thio-D-neuraminate.
3. A composition comprising an antigenic material and a compound of claim 1.
4. A composition according to claim 3 wherein the compound is present in an amount effective to exert an adjuvant effect.
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US05/965,559 US4229441A (en) 1978-12-01 1978-12-01 Immunologic adjuvant
AU53009/79A AU5300979A (en) 1978-12-01 1979-11-20 Derivatives obtained by condensing 6-(5-cholesten-3beta- yloxy) hexyl compound with hexoses
CA000340239A CA1147722A (en) 1978-12-01 1979-11-20 Immunologic adjuvant
EP79400932A EP0012083A1 (en) 1978-12-01 1979-11-29 Immunologic adjuvant, process for preparation and vaccine containing such adjuvant
JP15447979A JPS5589298A (en) 1978-12-01 1979-11-30 Immunological adjuvant
DK509579A DK509579A (en) 1978-12-01 1979-11-30 GLYCOLIPID COMPOUNDS, THEIR PREPARATION AND USE, AND INTERMEDIATES FOR USE IN THEIR PREPARATION

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US4621137A (en) * 1982-12-24 1986-11-04 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Alpha-glycosyl ginsenosides
US4652637A (en) * 1985-11-25 1987-03-24 Merck & Co., Inc. Steroidal glycolipids
US4652553A (en) * 1985-11-25 1987-03-24 Merck & Co., Inc. Steroidal glycolipids as host resistance stimulators
EP0224173A1 (en) 1985-11-25 1987-06-03 Merck & Co. Inc. Steroidal glycolipids as host resistance stimulators
US4942154A (en) * 1987-06-18 1990-07-17 Merck & Co., Inc. Steroidal glycolipids as host resistance stimulators against viral infection
US4970199A (en) * 1987-06-18 1990-11-13 Merck & Co., Inc. Steroidal glycolipids as host resistance stimulators against viral infection
US5019568A (en) * 1987-06-18 1991-05-28 Merck & Co., Inc. Steroidal glycolipids as host resistance stimulators against viral infection
US5380829A (en) * 1991-03-29 1995-01-10 The Nisshin Oil Mills, Ltd. Thioglycoside analogs of gangliosides
US5514784A (en) * 1993-03-19 1996-05-07 Wong; Chi-Huey Thio linked glycosyl compounds
US5650152A (en) * 1988-10-27 1997-07-22 Regents Of The University Of Minnesota Liposome immunoadjuvants containing IL-2
US5660855A (en) * 1995-02-10 1997-08-26 California Institute Of Technology Lipid constructs for targeting to vascular smooth muscle tissue
US5695738A (en) * 1995-06-15 1997-12-09 Glycomed Incorporated Steroidal C-glycosides
US6075132A (en) * 1998-01-08 2000-06-13 Bio Research Corporation Of Yokohama Ursodeoxycholic acid derivatives and methods for producing them
US6444649B1 (en) 1998-04-10 2002-09-03 Mitsubishi Chemical Corporation Solid dispersion containing sialic acid derivative
US6656481B1 (en) 1996-09-06 2003-12-02 Mitsubishi Chemical Corporation Vaccinal preparations
WO2004089969A2 (en) * 2003-04-02 2004-10-21 The Government Of The United States Of America As Represented By The Secretary, Department Of Healthand Human Services Cholesterol-containing compounds and their use as immunogens against borrelia burgdorferi

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US3585186A (en) * 1969-01-31 1971-06-15 American Cyanamid Co Preparation of glycopyranosiduronides and glycopyranosides and products resulting therefrom
US3752803A (en) * 1970-10-27 1973-08-14 Boehringer Sohn Ingelheim Derivatives of 3 - (3' beta-tridigitoxosyl-14' beta-hydroxy-5' beta-androstan - 17' beta-yl)-acrylic acid
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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4621137A (en) * 1982-12-24 1986-11-04 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Alpha-glycosyl ginsenosides
US4652637A (en) * 1985-11-25 1987-03-24 Merck & Co., Inc. Steroidal glycolipids
US4652553A (en) * 1985-11-25 1987-03-24 Merck & Co., Inc. Steroidal glycolipids as host resistance stimulators
EP0224173A1 (en) 1985-11-25 1987-06-03 Merck & Co. Inc. Steroidal glycolipids as host resistance stimulators
US4942154A (en) * 1987-06-18 1990-07-17 Merck & Co., Inc. Steroidal glycolipids as host resistance stimulators against viral infection
US4970199A (en) * 1987-06-18 1990-11-13 Merck & Co., Inc. Steroidal glycolipids as host resistance stimulators against viral infection
US5019568A (en) * 1987-06-18 1991-05-28 Merck & Co., Inc. Steroidal glycolipids as host resistance stimulators against viral infection
US5650152A (en) * 1988-10-27 1997-07-22 Regents Of The University Of Minnesota Liposome immunoadjuvants containing IL-2
US5380829A (en) * 1991-03-29 1995-01-10 The Nisshin Oil Mills, Ltd. Thioglycoside analogs of gangliosides
US5514784A (en) * 1993-03-19 1996-05-07 Wong; Chi-Huey Thio linked glycosyl compounds
US5660855A (en) * 1995-02-10 1997-08-26 California Institute Of Technology Lipid constructs for targeting to vascular smooth muscle tissue
US5695738A (en) * 1995-06-15 1997-12-09 Glycomed Incorporated Steroidal C-glycosides
US6656481B1 (en) 1996-09-06 2003-12-02 Mitsubishi Chemical Corporation Vaccinal preparations
US6075132A (en) * 1998-01-08 2000-06-13 Bio Research Corporation Of Yokohama Ursodeoxycholic acid derivatives and methods for producing them
US6444649B1 (en) 1998-04-10 2002-09-03 Mitsubishi Chemical Corporation Solid dispersion containing sialic acid derivative
WO2004089969A2 (en) * 2003-04-02 2004-10-21 The Government Of The United States Of America As Represented By The Secretary, Department Of Healthand Human Services Cholesterol-containing compounds and their use as immunogens against borrelia burgdorferi
WO2004089969A3 (en) * 2003-04-02 2004-12-16 Us Gov Health & Human Serv Cholesterol-containing compounds and their use as immunogens against borrelia burgdorferi
US20060204515A1 (en) * 2003-04-02 2006-09-14 Gil Ben-Menachem Cholesterol-containing compounds and their use as immunogens against borrelia burgdorferi
EP2030979A1 (en) * 2003-04-02 2009-03-04 The Government of the United States of America as Represented by The Department of Health and Human Services Cholesterol-containing compounds and their use as immunogens against borrelia burgdorferi
US7666846B2 (en) * 2003-04-02 2010-02-23 The United States Of America As Represented By The Department Of Health And Human Services Cholesterol-containing compounds and their use as immunogens against Borrelia burgdorferi
US20100062023A1 (en) * 2003-04-02 2010-03-11 The Government of the United States of America as represented by the Secretary, Department of Cholesterol-containing compounds and their use as immunogens against borrelia burgdorferi

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DK509579A (en) 1980-06-02

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